Effect of colonisation with Neisseria lactamica on cross-reactive anti-meningococcal B-cell responses: a randomised, controlled, human infection trial.

BACKGROUND: Pharyngeal colonisation by the commensal bacterium Neisseria lactamica inhibits colonisation by Neisseria meningitidis and has an inverse epidemiological association with meningococcal disease. The mechanisms that underpin this relationship are unclear, but could involve the induction of cross-reactive immunity. In this study, we aimed to evaluate whether colonisation with N lactamica induces N lactamica-specific B-cell responses that are cross-reactive with N meningitidis. METHODS: In this randomised, placebo-controlled, human infection trial at University Hospital Southampton Clinical Research Facility (Southampton, UK), healthy adults aged 18-45 years were randomly assigned (2:1) to receive intranasal inoculation with either 105 colony-forming units of N lactamica in 1 mL phosphate-buffered saline (PBS) or 1 mL PBS alone. Participants and researchers conducting participant sampling and immunological assays were masked to allocation. The primary endpoint was the frequency of circulating N lactamica-specific plasma cells and memory B cells after N lactamica inoculation (day 7-28) compared with baseline values (day 0), measured using enzyme-linked immunospot assays. The secondary endpoint was to measure the frequency of N meningitidis-specific B cells. In a second study, we measured the effect of duration of N lactamica colonisation on seroconversion by terminating carriage at either 4 days or 14 days with single-dose oral ciprofloxacin. The studies are now closed to participants. The trials are registered with ClinicalTrials.gov, NCT03633474 and NCT03549325. FINDINGS: Of 50 participants assessed for eligibility between Sept 5, 2018, and March 3, 2019, 31 were randomly assigned (n=20 N lactamica, n=11 PBS). Among the 17 participants who were colonised with N lactamica, the median baselines compared with peak post-colonisation N lactamica-specific plasma-cell frequencies (per 105 peripheral blood mononuclear cells) were 0·0 (IQR 0·0-0·0) versus 5·0 (1·5-10·5) for IgA-secreting plasma cells (p<0·0001), and 0·0 (0·0-0·0) versus 3·0 (1·5-9·5) for IgG-secreting plasma cells (p<0·0001). Median N lactamica-specific IgG memory-B-cell frequencies (percentage of total IgG memory B cells) increased from 0·0024% (0·0000-0·0097) at baseline to 0·0384% (0·0275-0·0649) at day 28 (p<0·0001). The frequency of N meningitidis-specific IgA-secreting and IgG-secreting plasma cells and memory B cells also increased signficantly in participants who were colonised with N lactamica. Upper respiratory tract symptoms were reported in ten (50%) of 20 participants who were inoculated with N lactamica and six (55%) of 11 participants who were inoculated with PBS (p>0·99). Three additional adverse events (two in the N lactamica group and one in the PBS group) and no serious adverse events were reported. In the second study, anti-N lactamica and anti-N meningitidis serum IgG titres increased only in participants who were colonised with N lactamica for 14 days. INTERPRETATION: Natural immunity to N meningitidis after colonisation with N lactamica might be due to cross-reactive adaptive responses. Exploitation of this microbial mechanism with a genetically modified live vector could protect against N meningitidis colonisation and disease. FUNDING: Wellcome Trust, Medical Research Council, and NIHR Southampton Biomedical Research Centre.

A recombinant commensal bacteria elicits heterologous antigen-specific immune responses during pharyngeal carriage.

The human nasopharynx contains a stable microbial ecosystem of commensal and potentially pathogenic bacteria, which can elicit protective primary and secondary immune responses. Experimental intranasal infection of human adults with the commensal Neisseria lactamica produced safe, sustained pharyngeal colonization. This has potential utility as a vehicle for sustained release of antigen to the human mucosa, but commensals in general are thought to be immunologically tolerated. Here, we show that engineered N. lactamica, chromosomally transformed to express a heterologous vaccine antigen, safely induces systemic, antigen-specific immune responses during carriage in humans. When the N. lactamica expressing the meningococcal antigen Neisseria Adhesin A (NadA) was inoculated intranasally into human volunteers, all colonized participants carried the bacteria asymptomatically for at least 28 days, with most (86%) still carrying the bacteria at 90 days. Compared to an otherwise isogenic but phenotypically wild-type strain, colonization with NadA-expressing N. lactamica generated NadA-specific immunoglobulin G (IgG)- and IgA-secreting plasma cells within 14 days of colonization and NadA-specific IgG memory B cells within 28 days of colonization. NadA-specific IgG memory B cells were detected in peripheral blood of colonized participants for at least 90 days. Over the same period, there was seroconversion against NadA and generation of serum bactericidal antibody activity against a NadA-expressing meningococcus. The controlled infection was safe, and there was no transmission to adult bedroom sharers during the 90-day period. Genetically modified N. lactamica could therefore be used to generate beneficial immune responses to heterologous antigens during sustained pharyngeal carriage.

Provision of Additional Cup Drinkers Mildly Alleviated Moderate Heat Stress Conditions in Broiler Chickens.

Sixty-four broilers of a commercial strain in a 2 × 2 factorial design with two environmental conditions: normal (N; 20°C, 50% RH) or MHS (30°C, 70% RH for 6 h/day for 3 consecutive days), and two drinker treatments; standard (B; a bell drinker/pen) or additional (A; as B + two cup drinkers). Each treatment had 16 birds with half of them implanted with temperature-ID chips to estimate the change in core body temperature (ΔCBT). Comb (CT) and feet (FT) temperatures were measured with atemperature probe, behavior was recorded with digital cameras, daily weight gain, feed intakes and litter moisture content (LMC) were also monitored. Data were subjected to General Linear Model (GLM) analysis. MHS broilers had greater ΔCBT, CT, and FT and more time spent panting and drinking compared to N broilers. Provision of additional cup drinkers reduced ΔCBT but enhanced heat loss from the comb and feet. At 6HS, MHS birds showed a tendency to spend more time drinking from the cup drinkers compared to N birds without compromising LMC. MHS suppressed feed intake and not weight gain.

Cell Proliferation in the Adult Chicken Hippocampus Correlates With Individual Differences in Time Spent in Outdoor Areas and Tonic Immobility.

Access to outdoor areas is provided as a means of enhancing welfare in commercial systems for laying hens (Gallus gallus domesticus), but substantial individual differences exist in their proportional use. Baseline cell proliferation levels of Adult Hippocampal Neurogenesis (AHN) have been associated with individual differences in reactive vs. proactive coping style, and in both mammals and birds, AHN is upregulated by positive experiences including environmental enrichment and exercise. We thus sought to explore whether individual differences in use of outdoor areas and in tonic immobility responses (indicative of fearfulness) were associated with hippocampal cell proliferation and neuronal differentiation. Radio frequency identification technology was used to track the ranging behavior of 440 individual focal hens within a commercially-relevant system over a 72-days period, after which tonic immobility durations were measured. Following hippocampal tissue collection from 58 focal hens, proliferation and neuronal differentiation were measured through quantitative PCR for proliferating cell nuclear antigen (PCNA) and doublecortin mRNA, respectively. Individual differences in tonic immobility duration positively correlated with PCNA expression over the whole hippocampal formation, while greater time spent in outdoor areas (the grassy range and stone yard) was associated with higher proliferation in the rostral subregion. Basal proliferation in the chicken hippocampal formation may thus relate to reactivity, while levels in the rostral region may be stimulated by ranging experience. Doublecortin expression in the caudal hippocampus negatively co-varied with time on the grassy range, but was not associated with tonic immobility duration. This suggests that ranging outside may be associated with stress. Within laying hen flocks, individual differences in hippocampal plasticity thus relate to coping style and use of external areas.

Influence of the Competition Context on Arousal in Agility Dogs.

To determine whether participation in dog agility has an impact on canine arousal and welfare, this study aimed firstly to identify the effect of the competition context on arousal changes experienced by dogs, as distinct from purely physical participation in agility, and secondly to assess the handlers' ability to recognize this. Behaviors indicative of changes in arousal were recorded for twenty dogs immediately before completion of both a competition and a training run, whilst the accuracy of handlers' observations of their dogs' behavior was examined via questionnaire. Whilst a moderate number of behaviors presented with greater frequency or duration in competition, the total number of different arousal behaviors performed was higher for dogs in competition (p < 0.01). Context had a relatively modest effect on the level of arousal of agility dogs, with a greater number of behaviors indicating increased arousal in competition. Such increased arousal may adversely influence the success of dog-handler partnerships in competition.  In both contexts, handlers observed fewer behaviors than their dogs performed and this finding may have implications for dog welfare.

Patterns of ovarian cancer and uterine cancer mortality and incidence in the contiguous USA.

The main objective is to investigate the geographical variation in ovarian cancer and uterine cancer mortality, and to test associations between some risk factors and these cancer types in the contiguous US for mortality and for incidence. The modern disease surveillance software SaTScan™ was used for a spatial cluster analysis to assess any observable geographical variation in ovarian and uterine cancer mortality rates and to identify and test for spatial clusters with elevated relative risk. The analyses were first completed using age adjusted cancer rates for ovarian cancer and for uterine cancer. The cancer data was then adjusted for the risk factors (or covariates) obesity rate, smoking rate, urban, poverty rate, college education rate, race, opioids mortality rate, and for arsenic intake from well water rate. All used data for cancer mortality were for 2000-2014 while incidence data were for 2011-2015. There exist seven significant mortality clusters of ovarian cancer, with large clusters in NW, NE and SE of the US, and there exist two large mortality clusters of uterine cancer in NE and Central US. Most risk factors studied for mortality and for incidence were significant at significance levels much lower than 0.05 for either of the two cancer types, except race for ovarian cancer mortality and arsenic for ovarian incidence. This study has identified several important factors, and these findings could be used for a more effective search for cancer prevention for uterine and ovarian cancer. LIMITATIONS OF THE STUDY: The accuracy of the data could not be controlled as data were downloaded from websites. While the mortality data was complete, the incidence data had counties with missing data. The data were obtained at the county resolution. No data were available on women who had one type of cancer and then had the second type of cancer later in life. Only purely spatial clusters were studied and no temporal analysis was done.

Elevated levels of the stress hormone, corticosterone, cause 'pessimistic' judgment bias in broiler chickens.

Pessimistic judgment biases, whereby humans or non-human animals interpret ambiguous information negatively, are hypothesised to be one of the suite of adaptive changes that comprise the vertebrate stress response. To test this hypothesis, we asked whether experimentally elevating levels of the glucocorticoid stress hormone, corticosterone, in broiler chickens produced a pessimistic judgment bias. We trained young chickens to discriminate a stimulus (paper cone) placed at two locations in an arena, one associated with reward (mealworms) and one with punishment (air puff). During seven days of non-invasive administration of either corticosterone or vehicle control, we tested the birds' responses to the cone placed at ambiguous locations between the trained locations. Corticosterone-treated birds were more likely than controls to respond as if punishment was likely when the cone was placed near to the punished location. The degree of this 'pessimism' was associated with smaller relative spleen weight, which is a documented consequence of chronic stress in chickens. We conclude that changes in corticosterone levels in chickens are sufficient to cause a specific change in decision making, dubbed 'pessimism', whereby corticosterone-treated birds showed an increased expectation of punishment in the face of ambiguous information. Pessimism could be a useful welfare indicator in chickens.

Epidemiological associations between brachycephaly and upper respiratory tract disorders in dogs attending veterinary practices in England.

BACKGROUND: Brachycephalic dog breeds are increasingly common. Canine brachycephaly has been associated with upper respiratory tract (URT) disorders but reliable prevalence data remain lacking. Using primary-care veterinary clinical data, this study aimed to report the prevalence and breed-type risk factors for URT disorders in dogs. RESULTS: The sampling frame included 170,812 dogs attending 96 primary-care veterinary clinics participating within the VetCompass Programme. Two hundred dogs were randomly selected from each of three extreme brachycephalic breed types (Bulldog, French Bulldog and Pug) and three common small-to medium sized breed types (moderate brachycephalic: Yorkshire Terrier and non-brachycephalic: Border Terrier and West Highland White Terrier). Information on all URT disorders recorded was extracted from individual patient records. Disorder prevalence was compared between groups using the chi-squared test or Fisher's test, as appropriate. Risk factor analysis used multivariable logistic regression modelling. During the study, 83 (6.9 %) study dogs died. Extreme brachycephalic dogs (median longevity: 8.6 years, IQR: 2.4-10.8) were significantly younger at death than the moderate and non-brachycephalic group of dogs (median 12.7 years, IQR 11.1-15.0) (P < 0.001). A higher proportion of deaths in extreme brachycephalic breed types were associated with URT disorders (4/24 deaths, 16.7 %) compared with the moderate and non-brachycephalic group (0/59 deaths, 0.0 %) (P = 0.001). The prevalence of having at least one URT disorder in the extreme brachycephalic group was higher (22.0 %, 95 % confidence interval (CI): 18.0-26.0) than in the moderate and non-brachycephalic group (9.7 %, 95 % CI: 7.1-12.3, P < 0.001). The prevalence of URT disorders varied significantly by breed type: Bulldogs 19.5 %, French Bulldogs 20.0 %, Pugs 26.5 %, Border Terriers 9.0 %, West Highland White Terriers 7.0 % and Yorkshire Terriers 13.0 % (P < 0.001). After accounting for the effects of age, bodyweight, sex, neutering and insurance, extreme brachycephalic dogs had 3.5 times (95 % CI: 2.4-5.0, P < 0.001) the odds of at least one URT disorder compared with the moderate and non-brachycephalic group. CONCLUSIONS: In summary, this study reports that URT disorders are commonly diagnosed in Bulldog, French Bulldog, Pug, Border Terrier, WHWT and Yorkshire Terrier dogs attending primary-care veterinary practices in England. The three extreme brachycephalic breed types (Bulldog, French Bulldog and Pug) were relatively short-lived and predisposed to URT disorders compared with three other small-to-medium size breed types that are commonly owned (moderate brachycephalic Yorkshire Terrier and non-brachycephalic: Border Terrier and WHWT).

Müller's muscle-conjunctival resection for upper eyelid ptosis: correlation between amount of resected tissue and outcome.

AIMS: To explore the relationship between the amount of resected Müller's muscle-conjunctiva (MMCR) and clinical outcome in patients undergoing upper eyelid ptosis surgery. METHODS: 49 patients underwent 87 MMCR surgeries. The total areas of the specimen and of MM were measured in pixels. RESULTS: The average percentage of muscle tissue in relation to total excised tissue was 21%. Intraoperative MMC tissue measurements and postoperative improvement in eyelid position (delta marginal reflex distance 1 (MRD1)) were positively correlated (R=0.427, p=0.09). There was a weak correlation between total areas measured on the histological slides and the intraoperative MMCR values (R=0.3, p=0.057). Total histological areas did not correlate with the delta change in eyelid position or with the amount and percentage of resected muscle tissue and the extent of improvement in eyelid position (delta MRD1) or final eyelid position (postoperative MRD1). CONCLUSIONS: Post-MMCR improvement in eyelid positions did not correlate with the percentage of MM in the excised tissue. We believe that the mechanism responsible for surgical outcome is plication or scarring of the posterior lamella and not the amount of resected MM. More lift in eyelid position can be anticipated when more tissue is excised by MMCR, and not when more muscle is excised.

The Effect of Steps to Promote Higher Levels of Farm Animal Welfare across the EU. Societal versus Animal Scientists' Perceptions of Animal Welfare.

Information about animal welfare standards and initiatives from eight European countries was collected, grouped, and compared to EU welfare standards to detect those aspects beyond minimum welfare levels demanded by EU welfare legislation. Literature was reviewed to determine the scientific relevance of standards and initiatives, and those aspects going beyond minimum EU standards. Standards and initiatives were assessed to determine their strengths and weaknesses regarding animal welfare. Attitudes of stakeholders in the improvement of animal welfare were determined through a Policy Delphi exercise. Social perception of animal welfare, economic implications of upraising welfare levels, and differences between countries were considered. Literature review revealed that on-farm space allowance, climate control, and environmental enrichment are relevant for all animal categories. Experts' assessment revealed that on-farm prevention of thermal stress, air quality, and races and passageways' design were not sufficiently included. Stakeholders considered that housing conditions are particularly relevant regarding animal welfare, and that animal-based and farm-level indicators are fundamental to monitor the progress of animal welfare. The most notable differences between what society offers and what farm animals are likely to need are related to transportation and space availability, with economic constraints being the most plausible explanation.

Immunisation with recombinant proteins subolesin and Bm86 for the control of Dermanyssus gallinae in poultry.

Dermanyssus gallinae has a worldwide distribution and is considered to be the most serious and economically significant ectoparasite affecting egg-laying poultry in Europe. Recombinant Bm86 and subolesin proteins derived from Boophilus microplus ticks and Aedes albopictus mosquitoes were used to immunise poultry in an attempt to control D. gallinaein vitro. Immunisation with subolesin and Bm86 stimulated different profiles of IgY response, whilst Bm86 but not subolesin was recognized by IgY on western blots. Orthologues for Bm86 were not found in D. gallinae by PCR, but a 150 bp fragment aligned with mammalian akirin 1 and a 300 bp fragment aligned with Amblyomma hebraeum were amplified by subolesin PCR. D. gallinae mortality after feeding was 35.1% higher (P=0.009) in the Subolesin group and 23% higher (not significant) in the Bm86 compared to the Control group. Thus it can be concluded that immunisation with recombinant subolesin can stimulate a protective response in laying hens against D. gallinae.

The poultry red mite (Dermanyssus gallinae): a potential vector of pathogenic agents.

The poultry red mite, D. gallinae has been involved in the transmission of many pathogenic agents, responsible for serious diseases both in animals and humans. Nowadays, few effective methods are available to control the ectoparasite in poultry farms. Consequently, this is an emerging problem which must be taken into account to maintain good health in commercial egg production. This paper addresses the vector capacity of the ectoparasite with special emphasis on salmonellae, pathogenic agents responsible for many of the most important outbreaks of food-borne diseases worlwide. It has been experimentally shown that D. gallinae could act as a biological vector of S. enteritidis and natural carriage of these bacteria by the mite on poultry premises has also been reported. It was also found that D. gallinae carried other pathogens such as E. coli, Shigella sp., and Staphylococcus, thus increasing the list of pathogenic agents potentially transmitted by the mite.

Endosymbiotic bacteria living inside the poultry red mite (Dermanyssus gallinae).

This study investigated the endosymbiotic bacteria living inside the poultry red mite collected from five samples of one commercial farm from the UK and 16 farms from France using genus-specific PCR, PCR-TTGE and DNA sequencing. Endosymbiotic bacteria are intracellular obligate organisms that can cause several phenotypic and reproductive anomalies to their host and they are found widespread living inside arthropods. The farm sampled from the UK was positive for bacteria of the genera Cardinium sp. and Spiroplasma sp. From France, 7 farms were positive for Cardinium sp., 1 farm was positive for Spiroplasma sp., 1 farm was positive for Rickettsiella sp. and 2 farms were positive for Schineria sp. However, it was not possible to detect the presence of the genus Wolbachia sp. which has been observed in other ectoparasites. This study is the first report of the presence of endosymbionts living inside the poultry red mite. The results obtained suggest that it may be possible that these bacterial endosymbionts cause biological modifications to the poultry red mite.

Characterization of the immune response of domestic fowl following immunization with proteins extracted from Dermanyssus gallinae.

Dermanyssus gallinae is the most significant ectoparasite of European poultry egg laying production systems due to high costs of control and associated production losses as well as adverse effects on bird welfare. In this study, soluble proteins were extracted from unfed D. gallinae (DGE) using a urea-based detergent and ultra-filtration, passed through a 0.22 microm filter and blended aseptically with adjuvant. One group of laying hens was immunized with DGE and adjuvant (Montanide ISA 50 V) whilst another group (Control) received physiological saline and adjuvant. All birds were immunized on two occasions, 21 days apart. Antibody response to immunization was determined by ELISA and western blotting using immunoglobulins (Igs) extracted from egg yolk. DGE immunization of hens resulted in a significant (P<0.05) IgY response compared to controls, although there was no significant difference in IgM response between treatments. A number of proteins were identified by western blotting using IgY antibodies from DGE immunized birds, most prominently at 40 and 230kDa. Analysis of proteins from approximately corresponding bands on SDS-PAGE confirmed the identity of tropomyosin, whilst other proteins showed high sequence homology with myosin and actin from other arachnid and insect species. Immunization of hens with DGE resulted in a 50.6% increase in mite mortality (P<0.001) 17h after feeding when tested by an in vitro mite feeding model. Data in this study demonstrate that somatic antigens from D. gallinae can be used to stimulate a protective immune response in laying hens. Further work is needed to identify other proteins of interest that could confer higher protection against D. gallinae, as well as optimization of the vaccination and in vitro testing protocol.

Variation in chemical composition and acaricidal activity against Dermanyssus gallinae of four eucalyptus essential oils.

The results of this study suggest that certain eucalyptus essential oils may be of use as an alternative to synthetic acaricides in the management of the poultry red mite, Dermanyssus gallinae. At a level of 0.21 mg/cm(2), the essential oil from Eucalyptus citriodora achieved 85% mortality in D. gallinae over a 24 h exposure period in contact toxicity tests. A further two essential oils from different eucalyptus species, namely E. globulus and E. radiata, provided significantly (P < 0.05) lower mite mortality (11 and 19%, respectively). Notable differences were found between the eucalyptus essential oils regarding their chemical compositions. There appeared to be a trend whereby the essential oils that were composed of the fewer chemical components were the least lethal to D. gallinae. It may therefore be the case that the complexity of an essential oil's chemical make up plays an important role in dictating the toxicity of that oil to pests such as D. gallinae.

Use of plant-derived products to control arthropods of veterinary importance: a review.

The use of synthetic products in veterinary pest management is becoming increasingly problematic. Issues, including pest resistance, product withdrawal, undesirable environmental persistence, and high mammalian toxicity associated with synthetic pesticides, are driving research to identify new pest management approaches. One approach employs the repellent/toxic effects of plant-derived products (PDPs). Several pesticides based on PDPs are already available in some areas of pest management. This review highlights instances in which such products have been used with success against pests of domestic animals, livestock, apiculture, and poultry.

Immunological control of the poultry red mite.

In the current study whole poultry red mite antigens were extracted and birds were immunized subcutaneously with either antigen in adjuvant (antigen group) or PBS in adjuvant (control group). Immune responses of birds following immunization were investigated by ELISA and Western blotting, while vaccine efficacy was assessed by feeding of red mites on birds. Immunized birds showed a significant (P < 0.05) increase in IgY titers after immunization compared to controls, while immunoglobulin A (IgA) and IgM did not change significantly. However, the antigen group had a generally higher increase in all immunoglobulin titers compared to the controls. Western blotting identified a number of protein bands at different molecular weights, although these were not different between treatments. PCR analysis of whole mite protein identified bacterial DNA that might have confounded immunological data. In addition, there was a trend toward reduced survival rate of red mites feeding on antigen-immunized birds, but the difference was not statistically significant compared to controls. This study demonstrates the potential for somatic red mite antigens to stimulate an antibody-mediated immune response, although this response did not confer protection to birds.

The poultry red mite Dermanyssus gallinae as a potential carrier of vector-borne diseases.

The poultry red mite Dermanyssus gallinae is an obligatory blood-sucking parasite that is considered to be one of the most important ectoparasites in the poultry industry, mainly because it is responsible for important economic losses, leads to a reduction of welfare of laying hens, and may pose a disease risk to humans. As a result of these problems, much of the current research on this parasite targets new methods of control. Less attention has been paid to the importance of D. gallinae as a carrier of vector-borne diseases. Some authors have mentioned the possible involvement of D. gallinae in the transmission (both in vitro and directly isolated from the mites) of viral and bacterial agents. Our research group has demonstrated the presence of Mycobacterium spp. within D. gallinae. DNA coding for Mycobacterium spp. was successfully amplified from unfed adult D. gallinae, larvae, and eggs by using reverse transcription-polymerase chain reaction targeting the 16S rRNA gene. The results have suggested the possible transovarial and transstadial transmission of pathogens by D. gallinae.